Inhibin was first identified as a gonad-derived regulator of pituitary FSH; however, it has subsequently been shown to be a tumour suppressor in the gonad and adrenal glands. Whereas non-malignant regions of human primary prostate carcinomas express inhibin alpha-subunit (INHA), malignant tissues lack INHA transcript and protein, which is consistent with epigenetic regulation of the inhibin alpha-subunit gene (INHA) promoter. This study investigated whether methylation of the INHA promoter was responsible for inactivation of INHA transcription and translation in the prostate cancer cell lines, LNCaP, DU145 and PC3. Methylation of the promoter was revealed by bisulphite genomic sequencing and use of inhibitors of methylation and histone deacetylation resulted in reactivation of the INHA transcription and translation. Significant (P<0.05) downregulation of a luciferase reporter gene downstream from a methylated INHA promoter compared with unmethylated INHA promoter occurred in vitro. The data demonstrate that promoter methylation is associated with downregulation of the INHA gene in prostate cancer cell lines, which is consistent with its tumour suppressive role. Therefore INHA has a significant role in prostate tumorigenesis.